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. Author manuscript; available in PMC: 2022 Mar 31.
Published in final edited form as: Life Sci. 2021 Oct 9;286:120044. doi: 10.1016/j.lfs.2021.120044

UVB-induced eIF2α phosphorylation in keratinocytes depends on decreased ATF4, GADD34 and CReP expression levels

Verónica A Bahamondes Lorca a,b,c, Bernardo D Bastidas Mayorg a,b, Lingying Tong a,b,*, Shiyong Wu a,b,*
PMCID: PMC8969208  NIHMSID: NIHMS1789314  PMID: 34637792

Abstract

Aim:

To elucidate the mechanism behind the sustained high levels of phosphorylated eIF2α in HaCaT cells post-UVB.

Main methods:

In this study, expression levels of the machinery involved in the dephosphorylation of eIF2α (GADD34, CReP and PP1), as well as the PERK-eIF2α-ATF4-CHOP, IRE1α/XBP1s and ATF6α signaling cascades, were analyzed by western blot and fluorescence microscope.

Key findings:

Our data showed that UVB induces the phosphorylation of eIF2α, which induces the translation of ATF4 and consequently the expression of CHOP and GADD34. Nevertheless, UVB also suppresses the translation of ATF4 and GADD34 in HaCaT cells via a p-eIF2α independent mechanism. Therefore, the lack of ATF4, GADD34 and CReP is responsible for the sustained phosphorylation of eIF2α. Finally, our data also showed that UVB selectively modifies PERK and downregulates ATF6α expression but does not induce activation of the IRE1α/XBP1s pathway in HaCaT cells.

Significance:

Novel mechanism to explain the prolonged phosphorylation of eIF2α post-UVB irradiation.

Keywords: Ultraviolet radiation B, eIF2α, ATF4, GADD34, Keratinocytes

1. Introduction

Ultraviolet (UV) B radiation is known for damaging the skin and increasing the chance of developing skin cancer. Upon UVB radiation, the phosphorylation of the α-subunit of the eukaryotic initiation factor 2 (eIF2α) in the Serine 51 is mainly mediated by the kinases general control nonrepressed 2 (GCN2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) [1,2]. The phosphorylation of eIF2α inhibits the global protein synthesis, which is a necessary response for inducing cell survival after stressor stimuli that deregulate the cellular homeostasis [3]. The inhibition of protein translation by phosphorylated eIF2α (p-eIF2α) is responsible for the activation of some pro-survival pathways such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) detected in the early phase (≤6 h) of post-UVB irradiation [4]. This global translation inhibition induced by p-eIF2 is highly regulated via negative feedback in which the proteins: growth arrest and DNA damage-inducible protein (GADD34), protein phosphatase 1 (PP1) and the constitutive repressor of eIF2α phosphorylation (CReP), play an important role in restoring the translation of proteins by dephosphorylating p-eIF2α [5-7].

Our previous studies have shown that the levels of p-eIF2α in HaCaT cells stay elevated post-irradiation [4,8]. It is known that p-eIF2α favors the translation of specific proteins that contain upstream open reading frames (uORF), such as the transcription factor Activating transcription factor 4 (ATF4) [9]. ATF4 induces the transcription of C/EBP- homologous protein (CHOP) [10,11]. The translation of CHOP is favored by p-eIF2α due to the presence of the inhibitory uORF within its 5′-untranslated region (5′UTR) [12]. Meanwhile, CHOP and ATF4 positively regulate the transcription of GADD34 [13,14], while high levels of p-eIF2α benefits the translation of this protein, since it also contains uORF within its 5′UTR which removes the inhibition established by p-eIF2α [15]. However, the mechanisms responsible for maintaining elevated levels of p-eIF2α post-UVB are not fully revealed. In this study, we examined the regulatory relations between eIF2α phosphorylation, ATF4 translation, and the expression of CHOP and GADD34 after UVB irradiation in HaCaT cells, which are responsible for maintaining the elevated levels of p-eIF2α observed previously in this cell line post-UVB. Additionally, we analyzed the response of the endoplasmic reticulum (ER)-stress sensors: PERK, inositol requiring enzyme 1α (IRE1α) and activating transcription factor 6α (ATF6α) [16] after UVB treatment. We observed that in HaCaT cells, UVB does not activate the unfolded protein response, instead it induces differential effects on each of the three ER-stress sensors.

2. Materials and methods

2.1. Cell culture

Human keratinocytes HaCaT cells (AddexBio, T0020001) and mouse embryonic fibroblast (MEF) cells were grown in Dulbecco's minimal essential medium (Corning™, Cellgro™, 10-013-CV) supplemented with 10% v/v fetal bovine serum and 1% v/v penicillin/streptomycin, at 37 °C with 5% CO2.

2.2. UVB irradiation

UVB was generated from a bench XX-15 series UV lamp (UVP Inc.) equipped with a 15 W tube (302 nm, UVP Inc.). After the lamp warmed up for at least 5 min the intensity of the UVB was calibrated using a UVP model UVX digital radiometer (UVP Inc.). 10 and 50 mJ/cm2 of UVB were used with a dose rate of 0.85 mW/cm2 per second. Prior to UVB radiation, the medium was removed from the cells and added back after radiation.

2.3. Drug treatments

MLN4924 (NEDD8-activating enzyme inhibitor, tested at 0.5, 1, and 2 μM, Cayman Chemical, # 15217), MG-132 (proteasome inhibitor, used at 5 μM, Millipore, # 474790), and Thapsigargin (SERCA inhibitor, used at 1 μM, Cayman Chemical, # 10522), were prepared on Dimethyl sulfoxide (DMSO, Millipore Sigma, D2438) and diluted in cell medium to their final concentrations prior to use. Tunicamycin (Tm, Calbiochem, # 654380) was diluted in cell medium to a final concentration of 0.1 μg/mL.

2.4. Western blot analysis

Radioimmunoprecipitation assay (RIPA) buffer (100 mM Tris-HCl, 2% v/v Triton X-100, 300 mM NaCl, 0.2% w/v SDS, 10 mM EDTA, and 1% w/v Sodium deoxycholate) with the proteases and phosphatases inhibitor mixture (Complete™, Mini, EDTA-free Protease Inhibitor Cocktail, Roche, 11836170001 and PhosSTOP™, Roche, 4906845001, respectively) was used to lyse the cells. Cells were scraped directly from the plates, pipetted, and incubated on ice for 30 min while vortexing every 5 min. The lysate was centrifuged at 12,000 rpm and 4 °C for 10 min and the supernatant was saved. Protein concentration was measured using the DC protein assay (Bio-Rad Laboratories, Inc) according to manufacturer's instructions. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane (BioTrace™ NT Nitrocellulose Membrane, pore size 0.2 μm, Pall Corporation, ID: 66485). The membrane was blocked in 5% w/v milk in Tris-buffered saline plus Tween 20 (TBST) for 1 h and then incubated overnight with the antibodies anti PERK (1:1000, # 3192), IRE1α (1:1000, # 3294), peIF2α (1:1000, # 9721), and ATF4 (1:1000, # 11815) from Cell Signaling Technology®; ATF6α (1:1000, sc-22799), eIF2α (1:1000, sc-133132), PPP1R15A/GADD34 (1:200, sc-373815), PP1 (1:1000, sc-7482), and β-actin (1:1500, sc-47778) from Santa Cruz Biotechnology and PPPlR15B/CReP (1:1000, # 14634-1-AP) from Proteintech. The membranes were washed and then incubated with the corresponding HRP-conjugated-secondary antibody for 1 h at room temperature (Cell Signaling Technology®) in 5% w/v milk in TBST. Next, the membranes were developed using the West Pico Super Signal chemiluminescent substrate (Thermo Fisher Scientific, 34580) or the Western Sure PREMIUM Chemiluminescent Substrate (LI-COR Biosciences, 926-95000). Images were obtained from Odyssey® Fc Imaging System (LI-COR Biosciences) and quantified using the software Image Studio Lite (LI-COR Biosciences).

2.5. Immunofluorescence staining of ATF4 and ATF6α

Cells were fixed with paraformaldehyde (4% ν/v) for 10 min. at room temperature and then washed with PBS 1×. Next, the samples were permeabilized with 0.1% Triton X-100 in PBS 1× for 10 min. on ice. After washing them with PBS, cells were blocked with 5% w/v BSA for 1 h at room temperature. Cells were incubated with the primary antibodies against ATF4 (1:100, # 11815) and ATF6α (1:1000, sc-22799) overnight at 4 °C. After washing with PBS, cells were incubated with a secondary antibody anti rabbit (1:200, Vector laboratories) plus DAPI (4′-6-diamidin-2′-phenylindol-dihydrochlorid) for 1 h at room temperature. Pictures were taken using a fluorescence microscope (Axio Observer, Zeiss).

2.6. Reverse transcription polymerase chain reaction (RT-PCR)

HaCaT cells were irradiated with UVB at 10 and 50 mJ/cm2. RNA extraction was performed at 1, 3, and 6 h post irradiation. HaCaT cells without irradiation were used as negative control. For XBP1 analysis, total RNA was extracted by TRIzol reagent according to the manufacturer's protocol (15526096, Invitrogen). First-strand cDNA synthesis was reverse transcribed from 1 μg of RNA using Superscript IV reverse transcriptase (18090050, Invitrogen) following the manufacturer's protocol. PCR was performed using GoTaq® Green Master Mix, 2× (M712, Promega). Primers sequences (from Invitrogen) of the target XBP-1 were designed to recognize both, human XBP1u (NM 005080.3) and XBP1s (NM 001079539.1), (Forward) 5′-TTAAGA-CAGCGCTTGGGGATGGAT-3′, (Reverse) 5′-AGATGTTCTGGAGGGGT-GACAACT-3′ were used. The PCR was performed with the following protocol: 95 °C for 2 min; 30 cycles at 95 °C for 30 s, 63 °C for 30 s, 72 °C for 30 s and finally, a cycle of 5 min at 72 °C. Data was analyzed after electrophoresis on a 4% agarose gel on 1× TBE buffer. For GADD34, ATF6α, ATF4 and CHOP mRNA analysis total RNA was extracted by the RNeasy Mini Kit (74104, Qiagen) according to manufacturer's protocol. First-strand cDNA synthesis was reverse transcribed from 1 μg of RNA using Superscript IV reverse transcriptase (18090, Invitrogen) following the manufacturer's protocol. PCR was performed using Maxima SYBR Green/Fluorescein qPCR Master Mix (2×) (K0241, Thermo Scientific™). XBP1, ATF4, ATF6α, and GADD34 primers were designed using the program “AmplifX 1.4 [1.4.0] by Nicolas Jullien; Aix-Marseille Univ, CNRS, INP, Inst Neurophysiopathol, Marseille, France: https://inp.univ-amu.fr/en/amplifx-manage-test-and-design-your-primers-for-pcr”.

Primers sequences (from Invitrogen) of the targets were:

ATF4 (Forward) 5′-TGGTGAGTGCAAAGAGCTGGAA-3′

ATF4 (Reverse) 5′-ACAAGCACATTGACGCTCCTGA-3′

CHOP (Forward) 5′-CAGAACCAGCAGAGGTCACA-3′ [17]

CHOP (Reverse) 5′-AGCTGTGCCACTTTCCTTTC-3′ [17]

ATF6 (Forward) 5′-ATATTAATCACGGAGTTCCAGGGA-3′

ATF6 (Reverse) 5′-GTGAAATAACCGAGTTCAGCAAAG-3′

GADD34 (Forward) 5′-CTGATAAGAACCCAGGGGAGGA-3′

GADD34 (Reverse) 5′-ATCCTGGAGACAAGGCAGAAGTA-3′

GAPDH (Forward) 5′-TGCACCACCAACTGCTTAGC-3′ [4]

GAPDH (Reverse) 5′-GGCATGGACTGTGGTCATGAG-3′ [4]

β-actin (Forward) 5′-CACTCTTCCAGCCTTCCTTCC-3′ [4]

β-actin (Reverse) 5′-CGGACTCGTCATACTCCTGCT-3′ [4]

The qPCR was performed with the following protocol: 50 °C for 2 min, 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s. In order to check the quality of the PCR reaction at the end of each qPCR, the melting curve analysis was carried out, and the products of the PCR reaction were separated by electrophoresis on a 4% agarose gel on 1× TBE buffer. GADD34, ATF6α, ATF4 and CHOP data were analyzed using the relative expression ratio method [18].

2.7. Statistical analysis

Each experiment was repeated at least three times. The bar plots are expressed as the geometric mean values ± S.E. The significance of the differences between mean values was assessed using Student's t-test. p-Values smaller than 0.05 were considered significant.

3. Results

3.1. eIF2α phophorylation-ATF4-CHOP signaling cascade is activated in time- and dose-dependent manners post-UVB

The dose- and time-dependent effects of UVB on the phosphorylation of eIF2α (p-eIF2α) and the protein expression of the downstream factors ATF4 and CHOP were determined by western blot analysis (Fig. 1A). Our data shows that an increase of p-eIF2α is detected in a dose-dependent manner at 1 h post-UVB (10 and 50 mJ/cm2) (Fig. 1A, Lane 3 vs. 2 vs. 1). While p-eIF2α levels show no change at 3 and 6 h after 10 mJ/cm2 of UVB radiation (Fig. 1A, Lanes 4, 6 vs. 2), it increased in a time-dependent manner after 50 mJ/cm2 of UVB (Fig. 1A, Lane 7 vs 5 vs. 3). An increase of ATF4 is also detected at 1 h post-UVB (10 and 50 mJ/cm2) (Fig. 1A, Lane 3 vs. 2 vs. 1). The expression of ATF4 is further increased at 3 h and then decreased at 6 h after 10 mJ/cm2 UVB exposure (Fig. 1A, Lane 6 vs. 4 vs. 2). However, it did not changed between 1 and 3 h after 50 mJ/cm2 of UVB treatment (Fig. 1A, Lane 3 vs. 5), but then decreased at 6 h post irradiation (Fig. 1A, Lane 7 vs. 5 and vs. 3). An increase on CHOP was also detected in a dose- and time-dependent manner at 3 and 6 h post-UVB 10 mJ/cm2 (Fig. 1A, Lane 6 vs. 4 vs.1). While CHOP also increased at 3 and 6 h post UVB after 50 mJ/cm2 of UVB irradiation (Fig. 1A, Lanes 5, and 7 vs. 1), no further changes were observed between 3 and 6 h post irradiation (Fig. 1A, Lane 7 vs. 5). Our data shows that CHOP expression increased more after 10 mJ/cm2 than after 50 mJ/cm2 of UVB treatment (Fig. 1 A, Lanes 4 vs. 5; 6 vs. 7), which correlates to the protein levels of ATF4 seen in Fig. 1A. Additionally, we observed that after 50 mJ/cm2 of UVB HaCaT cells have a constant elevation of p-eIF2α, which does not correlate with the observed protein levels of ATF4 since at 6 h post UVB, p-eIF2α continued increasing while ATF4 almost returned to its background levels (Fig. 1A, Lanes 7, 5, 3 vs. 1).

Fig. 1.

Fig. 1.

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Evaluation of the p-eIF2α/ATF4/CHOP signaling cascade in HaCaT cells post UVB (10 and 50 mJ/cm2). (A) Representative western blot showing the protein levels of the p-eIF2α, eIF2α, ATF4 and CHOP. (B and C) Relative ATF4 and CHOP mRNA levels at 1, 3, and 6 h post UVB. *: p < 0.05. (D) Immunostaining of ATF4 (green) in HaCaT at 1, 3, and 6 h post UVB 50 mJ/cm2, positive control with Tg (1 μM, incubation time 2 h). Bar 20 μm, nuclei were stained with DAPI (red). (E) Western blot showing the protein levels of p-eIF2α and ATF4 in wild type (WT) and in non-phosphorylable eIF2α (S51A) mouse embryonic fibroblast cells (MEF).

The effects of UVB on the mRNA levels of ATF4 and CHOP were determined by qPCR analysis (Fig. 1B and C). Our data shows that ATF4 mRNA levels are increased at 1 h post-UVB at a dose-dependent manner (Fig. 1B, Bar 3 vs. 2 vs. 1), and then remain at the same level for 10 mJ/cm2 UVB (Fig. 1B, Bar 2 vs. 4, 6) or are non-statistically significantly decreased for 50 mJ/cm2 UVB (Fig. 1B, Bar 3 vs. 5, 7) at 3 and 6 h post-UVB. The CHOP mRNA levels are increased in a time- and dose-dependent manner at 1, 3, and 6 h post-UVB (10 and 50 mJ/cm2) (Fig. 1C). In addition, the increased expression of ATF4 was nuclear (Fig. 1D) indicating that it is activated after UVB exposure. This activation was also observed in others cell lines such as MEF (Fig. 1E, Line 2 versus 1) and in primary human adult keratinocytes (not shown), indicating it is not cell line specific. The existence of a p-eIF2α independent mechanism of regulation of ATF4 protein levels post-UVB was confirmed using wild type (WT) and non-phosphorylable (S51S) MEF cells, in which the lack of phosphorylation on eIF2α avoids the increase of ATF4 after UVB (Fig. 1E, Lane 4 vs. 2) which, in addition, decreases post-UVB (Fig. 1E, Lane 4 vs. 3). These results indicate that UVB induces the phosphorylation of eIF2α; as well as the expression of ATF4 and CHOP. However, the level of eIF2α phosphorylation is not always correlated with the expression levels of ATF4 and CHOP in HaCaT cells after UVB irradiation.

3.2. Sustained phosphorylation of eIF2α after UVB depends on the lack of ATF4, GADD34 and CReP

To analyze the potential reciprocal regulation of eIF2α phosphorylation from the downstream factors of eIF2-ATF4-CHOP signaling cascade, we evaluated the expressions of GADD34 and its binding protein PP1, which dephosphorylates eIF2α [5-7]. The expression of CReP, a constitutive counterpart of GADD34, was also analyzed. Our data shows that the expression of GADD34 is either decreased (Fig 2A, Lanes 2, 4, 6 vs. 1) or almost eliminated (Fig. 2A, Lanes 3, 5, 7 vs. 1) after 10 or 50 mJ/cm2 UVB exposure, respectively. The protein levels of PP1 show no detectable change (Fig. 2A, Lanes 2–7 vs. 1) and the expression of CReP is decreased, but not as significant as GADD34 post-UVB (Fig. 2A, Lanes 2–7 vs. 1). The loss of GADD34 after 50 mJ/cm2 UVB exposure is not due to the loss of the transcripts because GADD34 mRNA is increased after the exposure (Fig. 2B, Columns 2–4 vs. 1).

Fig. 2.

Fig. 2.

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Evaluation of the expression of proteins involved in the dephosphorylation of p-eIF2α post UVB (10 and 50 mJ/cm2) (A) Representative western blot showing the protein levels of GADD34, CReP, and PP1. (B) Relative GADD34 mRNA levels at 1, 3, and 6 h post UVB 50 mJ/cm2 *: p < 0.05.

To determine whether ATF4 protein levels after UVB are related to the phosphorylation levels of eIF2α, we inhibited the degradation of ATF4 by using the proteasome inhibitor MG132 (5 μM) or the NEDD8-activating enzyme (NAE) inhibitor MLN4924 (0.5, 1, and 2 μM) [19-21]. Our data shows that both treating with MG132 and MLN4924 increases ATF4 levels as well as GADD34 and CReP levels (Fig. 3A, Lane 4 vs. 3 and 2, Lane 6 vs. 5 and 2, and Line 8 vs. 7 and 2; and Fig. 3B, Lanes 6–8 vs. 5 and 1–4, and Lines 10–12 vs 9 and 1–4). The increased ATF4 levels correlates to a decreased phosphorylation level of eIF2α at 3 and 6 h post-UVB (50 mJ/cm2) (Fig. 3A, Lanes 6 vs. 5 and 8 vs. 7; and Fig. 3B, Lanes 6–8 vs. 5, and 10–12 vs. 9). Combined, these results suggest that the prolonged elevation of eIF2α phosphorylation in HaCaT cells might be due to the low inducibility of ATF4 after 50 mJ/cm2 of UVB irradiation.

Fig. 3.

Fig. 3.

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Relation between ATF4, GADD34 and p-eIF2α. (A) Representative western blot showing the protein levels of p-eIF2α, ATF4, GADD34 and CReP at 1, 3, and 6 h post UVB 50 mJ/cm2 in the presence or absence of the proteasome inhibitor MG132. (B) Representative western blot showing the protein levels of p-eIF2α, ATF4, GADD34 and CReP at 3 and 6 h post UVB 50 mJ/cm2 and after inhibiting the degradation of ATF4 and CReP by using the NAE inhibitor MLN4924 at different concentrations.

3.3. UVB induces differential effects on each ER-stress sensor in keratinocytes

In addition to the PERK-eIF2 cascade, two other ER-stress sensor - ATF6α and IRE1α - are also known to induce CHOP expression [22-28]. Thus, we determined the protein expression levels and migration patterns of all three sensors PERK, ATF6α and IRE1α using western blot analysis. Our data shows that while the expression of PERK is not affected, the PERK bands are up shifted after UVB exposure (50 mJ/cm2) (Fig. 4A, Lanes 3, 5, 7 vs. 1, 2, 4, 6). The band-shift post-UVB is approximately 50% less than the band-shift after thapsigargin (Tg) treatment (Fig. 4A, Lane 3, 5, 7 vs. 8). The expression of ATF6α is decreased in time- and dose-dependent manners after UVB irradiation (Fig. 4B, Lanes 1–9). However, unlike the tunicamycin (Tm) treated sample, in which the glycosylation of ATF6α is inhibited (Fig. 4B, Full-G) and ATF6α is activated (Fig. 4B, Cleaved), the cleaved band that corresponds to the nuclear ATF6α is not detectable after UVB treatment (Fig. 4B, Lanes 2–9 vs. 10). Surprisingly, while IRE1α is up shifted after Tg treatment (Fig. 4C, Lane 8), neither the expression nor the migration is changed for IRE1α after UVB exposure (Fig. 4C, Lanes 2–7). To further confirm that UVB has no effect on IRE1α, the alternative splicing of the XBP1 mRNA, a substrate of activated IRE1α [29] was evaluated. Our data shows that compared to the effect induced by Tg, in which XBP1u and XBP1s are observed, XBP1s is not induced in the HaCaT cell line after UVB irradiation (Fig. 4D, Lanes 2–9 vs. 10). These results corroborate that UVB selectively modifies PERK, downregulates ATF6α expression, but does not induce activation of the IRE1α/XBP1s pathway in HaCaT cells.

Fig. 4.

Fig. 4.

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Evaluation of the PERK, IRE1α and ATF6α protein levels in HaCaT cells post UVB (10 and 50 mJ/cm2) via SDS-PAGE. (A) Representative western blot showing no changes in the protein levels of PERK, but changes in its migration pattern after UVB (50 mJ/cm2). (B) Protein levels of ATF6α at 1, 3, and 6 h after UVB (10 and 50 mJ/cm2). Full: full-length ATF6α, Full-G: unglycosylated full-length ATF6α, cleaved: nuclear ATF6α, which is only visible after treatment with the positive control Tunicamycin. Bands between full and cleaved ATF6α are described as partially cleaved ATF6α. (C) Representative western blot showing no changes in the protein levels of IRE1α, as well as no changes in its migration pattern at different times after UVB (10 and 50 mJ/cm2). (D) Agarose gel showing the PCR products for XBP1u and XBP1s, the latter being induced only after treatment with the positive control Thapsigargin (Tg, 1 μM).

3.4. UVB decreased the levels of full length ATF6α in HaCaT cells

To determine if ATF6α is truly not activated and ATF6 reduction is due to a decrease on transcripts and/or protein degradation, we analyzed ATF6α protein expression and location in a time-dependent manner post-UVB (50 mJ/cm2). Our data shows that in control keratinocytes, ATF6α is located mainly around the nucleus, corresponding to the ATF6α located in the ER (Fig. 5A, white arrows on left column). While ATF6α is detected inside the nuclei of the HaCaT cells treated with Tg (Fig. 5A, right column), it significantly decreases in both the ER and nuclei at 6 h post-UVB 50 mJ/cm2 (Fig. 5A, Middle Column). This data confirms that, first, there is a decreased expression of ATF6α in HaCaT cells post-UVB, and second, the ER-stress-ATF6α pathway is not defected in the cells since higher levels of the ATF6α signal is present inside the nuclei after Tg (Fig. 5A, Right Column). To determine if the decrease of ATF6α is due to its expression -inhibition, or its degradation, we inhibited its proteasomal degradation (Fig. 5B). Our results show that MG132 (5 μM) increases the levels of ATF6α in both control and UVB irradiated HaCaT cells (Fig. 5B, even vs. odd Lines). However, the observed protein levels of ATF6α after UVB are lower in comparison to the control samples (Fig. 5B, Lines 3–8 vs. 1–2). In addition, a decrease of ATF6α mRNA levels is also detected at 6 h post-UVB 50 mJ/cm2 (Fig. 5C, UVB vs. control). These results indicate that in HaCaT cells, UVB decreases the expression of ATF6α without activating it; and both transcription reduction and protein degradation might contribute to the ATF6α reduction.

Fig. 5.

Fig. 5.

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Evaluation of the ATF6α pathway activation in HaCaT cells post UVB (10 and 50 mJ/cm2). (A) Immunostaining of ATF6α in HaCaT cells at 6 h post UVB. Controls cells show ATF6α located mainly around the nuclei (ER), while UVB decreases ATF6α protein levels. Positive control Thapsigargin induces the translocation of ATF6α to the nuclei. ATF6α (green), nuclei (red), bar 20 μm. (B) ATF6α partial recovery after proteasome inhibition by MG132 (5 μM) in UVB irradiated HaCaT cells. (C) Relative ATF6α mRNA levels at 1, 3, and 6 h post UVB 50 mJ/cm2. *: p < 0.05.

4. Discussion

Previous studies indicated that UV, including UVB and UVC, induces the phosphorylation of eIF2α in dose- and time-dependent manners; and cNOS-mediated activation of both PERK and GCN2 lead to the phosphorylation [8]. In this study, we determined the reciprocal effect of the downstream factors on eIF2α phosphorylation as well as the effect of UVB on PERK and other ER sensors. Our data shows that UVB induces eIF2α phosphorylation as well as ATF4 expression and activation (Fig. 1A and D), which does not agree with previous reports, which suggest that UV does not induce ATF4 expression [30,31]. The disagreement could be due to the differences in the time of analysis and/ or be cell line dependent. In fact, we observed that the peaks of ATF4 expression post-UVB are at different time intervals in three evaluated cell lines (HaCaT, MEF and HEKa cells, not shown). Our data further shows that the increased phosphorylation of eIF2α does not correlate with an increase of ATF4 expression (Fig. 1A) as previous reported that eIF2α phosphorylation translationally up-regulates the expression of ATF4 [9]. The unexpectedly low expression of ATF4 along with high p-eIF2α levels are not due to the loss of ATF4 mRNA as its transcription is not decreased, but rather increased, at 1–6 h post-UVB (Fig. 1B). The increased transcription of ATF4 was confirmed by treating the cells with actinomycin D, which diminished the increased ATF4 mRNA post-UVB (data not shown), ruling out the possibility of an increased stability of the mRNA. Since the phosphorylation of eIF2α decreases the overcharge of the ER by inhibiting the global protein translation and also activates cellular pathways that allow the cells to recover the proper homeostasis [3,16], the increase on p-eIF2α levels after a stressor stimulus such as UVB on HaCaT cells is intended to keep the cells alive. This agrees with our previous report in which the pro-survival pathway NF-κB induced at early time post-UVB (within 6 h), was activated through a non-canonical mechanism dependent of the translational inhibition induced by p-eIF2α [4]. However, these anti-apoptotic responses are still balanced by proapoptotic signaling pathways already described post-UVB [32,33].

Since the degradation of ATF4 does not fully explain the reduced levels of this protein (Fig. 1A), the presence of other mechanisms independent of the phosphorylation levels of eIF2α (as suggested by in Fig. 1E) needs to be involved in controlling the translation of ATF4. For example, proteins such as DEAD-box helicase 3 X-linked (DDX3), eukaryotic release factor 3a (eRF3a), and mTORC1 have been described to be involved in regulate the translation of ATF4 via mechanisms that are dependent or independent of p-eIF2α [34-37]. These mechanisms of regulation involve other members of the eukaryotic initiation factor family, such as eIF3, and eIF4F complex, which are known to be involved in the formation of stress granules and control the translation of specific proteins after various stimuli [34,36-42].

A similar mechanism of regulation could also explain the observed low protein levels of GADD34 (Fig 2A). This is also an unexpected result, because GADD34 expression was anticipated to increase due to: 1) the presence of uORFs in its 5′UTR region [13,43], 2) the increased expression of CHOP [5] after 3 and 6 h post UVB (Fig. 1A), and 3) the observed increased mRNA levels of GADD34 (Fig. 2B). Furthermore, our results show that the sum of the repression of ATF4 and GADD34 post UVB is a strong contributor to the high levels of p-eIF2α observed in HaCaT cells (Fig. 3). Additionally, UVB decreases the levels of CReP (Fig 2A), the constitutive counterpart of GADD34 that also controls the levels of p-eIF2α by inducing its dephosphorylation; this fact then could be maximizing the elevated levels of phosphorylated eIF2α post-UVB.

In this study, we also analyzed the expression of CHOP, a transcriptional factor which expression is regulated by ATF4 [10,11] and by a uORF within its 5′UTR [12]. As expected, the protein levels of CHOP increased post-UVB and correlate positively with the protein levels of ATF4 (Fig. 1A), however its mRNA levels increased in a time- and dose-dependent manner at 1, 3, and 6 h after 10 and 50 mJ/cm2 (Fig. 1C). Since the ATF6α and IRE1α pathways of the ER stress are also known to regulate CHOP expression [22-28], we further analyzed the activation status of these sensors (Fig. 4). Our data showed that only the PERK pathway suggests activation by UVB since neither the IRE1α/XBP1 nor the ATF6α pathways showed signs of activation (Fig. 4). Moreover, we observed that UVB decreases the levels of the full length ATF6α in a dose dependent mechanism, a decrease that was neither related to its activation, nor by an accelerate protein degradation, but rather because the decreased expression of the full length ATF6α (Figs. 4B and 5). These results indicate that UVB selectively induces changes in PERK and ATF6α without activating the general unfolding protein response.

5. Conclusion

Based on our results, we propose a signaling circuit as shown in Fig. 6. In HaCaT cells, UVB induces the phosphorylation of eIF2α via the kinases PERK and GCN2, as described previously [1,2]. The phosphorylation of eIF2α favors the translation of all the mRNAs on ATF4-CHOP-GADD34 signaling cascade. Lastly, GADD34, along with CReP, is responsible for the dephosphorylation of p-eIF2α, a step necessary to restart the global synthesis of proteins. However, even UVB slightly increases the mRNA of ATF4 by an unknown mechanism; it also inhibits the translation of ATF4 in a mechanism that is independent of p-eIF2α. In addition, UVB inhibits the translation of GADD34 (potentially by a mechanism similar to the one decreasing ATF4) and reduces the expression of CReP, which is responsible for maintaining the elevated levels of p-eIF2α post-irradiation. Then, the sustained high levels of p-eIF2α (due to the low levels of ATF4 and GADD34) on HaCaT cells post irradiation have a protective role which seeks to avoid the UVB-induced cell death. In addition, our results also indicate that UVB decreases the transcription and/or translation of ATF6α by an unknown mechanism, without inducing the activation of the ER sensor.

Fig. 6.

Fig. 6.

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Proposed model of the signaling circuit regulating the phosphorylation of eIF2α and the protein levels of ATF6α in HaCaT cells after UVB irradiation.

Acknowledgement

We would like to thank Ms. Evelyn Potter and Ms. Rachel Beha for her editorial assistance in preparing this manuscript. This work was partially supported by NIH CA086928 and ES030425 (to S Wu) and a graduate student scholarship from the Department of Chemistry and Biochemistry of Ohio University (to Bernardo D. Bastidas Mayorga).

Footnotes

CRediT authorship contribution statement

Verónica A. Bahamondes Lorca: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – original draft. Bernardo D. Bastidas Mayorga: Investigation, Validation, Formal analysis. Lingying Tong: Conceptualization, Methodology, Supervision, Project administration, Funding acquisition. Shiyong Wu: Conceptualization, Validation, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition.

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